INTERNATIONAL STANDARD Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Salmonella spp docx

The harmonization of test methods cannot be immediate, and for certain groups of products International Standards and/or national standards may already exist that do not comply with this

Reference numberISO 6579:2002(E)

Fourth edition2002-07-15

Microbiology of food and animal feeding stuffs — Horizontal method for the

detection of Salmonella spp

Microbiologie des aliments — Méthode horizontale pour la recherche des

Salmonella spp

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Contents Page

Foreword iv

Introduction v

1 Scope 1

2 Normative references 1

3 Terms and definitions 1

4 Principle 2

4.1 General 2

4.2 Pre-enrichment in non-selective liquid medium 2

4.3 Enrichment in selective liquid media 2

4.4 Plating out and identification 2

4.5 Confirmation of identity 2

5 Culture media, reagents and sera 3

5.1 General 3

5.2 Culture media and reagents 3

5.3 Sera 4

6 Apparatus and glassware 4

7 Sampling 5

8 Preparation of test sample 5

9 Procedure (see diagram in annex A) 5

9.1 Test portion and initial suspension 5

9.2 Non-selective pre-enrichment 6

9.3 Selective enrichment 6

9.4 Plating out and identification 6

9.5 Confirmation 6

10 Expression of results 10

11 Test report 10

12 Quality assurance 11

Annex A (normative) Diagram of procedure 12

Annex B (normative) Composition and preparation of culture media and reagents 14

Annex C (informative) Results of interlaboratory trial 24

Bibliography 27

Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies) The work of preparing International Standards is normally carried out through ISO technical committees Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3

The main task of technical committees is to prepare International Standards Draft International Standards adopted

by the technical committees are circulated to the member bodies for voting Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote

Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights ISO shall not be held responsible for identifying any or all such patent rights

ISO 6579 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology

This fourth edition cancels and replaces the third edition (ISO 6579:1993), which has been technically revised Annexes A and B form a normative part of this Interntional Standard Annex C is for information only

Introduction

Because of the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain products In this case, different methods, which are specific to these products, may be used if absolutely necessary for justified technical reasons Nevertheless, every attempt should be made to apply this horizontal method as far as possible

When this International Standard is next reviewed, account will be taken of all information then available regarding the extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of particular products

The harmonization of test methods cannot be immediate, and for certain groups of products International Standards and/or national standards may already exist that do not comply with this horizontal method It is hoped that when such standards are reviewed they will be changed to comply with this International Standard so that eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons

Microbiology of food and animal feeding stuffs — Horizontal

method for the detection of Salmonella spp

WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for detecting

Salmonella, and especially Salmonella Typhi and Salmonella Paratyphi, are only undertaken in properly

equipped laboratories, under the control of a skilled microbiologist, and that great care is taken in the disposal of all incubated materials

1 Scope

This International Standard specifies a horizontal method for the detection of Salmonella, including Salmonella Typhi and Salmonella Paratyphi

Subject to the limitations discussed in the Introduction, this International Standard is applicable to

 products intended for human consumption and the feeding of animals;

 environmental samples in the area of food production and food handling

WARNING — The method may not recover all Salmonella Typhi and Paratyphi

The following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard For dated references, subsequent amendments to, or revisions of, any of these publications do not apply However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below For undated references, the latest edition of the normative document referred to applies Members of ISO and IEC maintain registers of currently valid International Standards

ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and

decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension and decimal dilutions

ISO 7218:1996, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations ISO 8261, Milk and milk products — General guidance for the preparation of test samples, initial suspensions and

decimal dilutions for microbiological examination

3 Terms and definitions

For the purposes of this International Standard, the following terms and definitions apply

3.1

Salmonella

microorganisms which form typical or less typical colonies on solid selective media and which display the biochemical and serological characteristics described when tests are carried out in accordance with this International Standard

3.2

detection of Salmonella

determination of the presence or absence of Salmonella (3.1), in a particular mass or volume of product, when

tests are carried out in accordance with this International Standard

4 Principle

4.1 General

The detection of Salmonella necessitates four successive stages (see also annex A)

NOTE The Salmonella may be present in small numbers and are often accompanied by considerably larger numbers of

other Enterobacteriaceæ or other families Furthermore, pre-enrichment is necessary to permit the detection of low numbers of

Salmonella or injured Salmonella

4.2 Pre-enrichment in non-selective liquid medium

Buffered peptone water is inoculated at ambient temperature with the test portion, then incubated at 37 °C ± 1 °C for 18 h ± 2 h

For certain foodstuffs the use of other pre-enrichment procedures is necessary See 9.1.2

For large quantities, the buffered peptone water should be heated to 37 °C ± 1 °C before inoculation with the test portion

4.3 Enrichment in selective liquid media

Rappaport-Vassiliadis medium with soya (RVS broth) and Muller-Kauffmann tetrathionate/novobiocin broth (MKTTn broth) are inoculated with the culture obtained in 4.2

The RVS broth is incubated at 41,5 °C ± 1 °C for 24 h ± 3 h, and the MKTTn broth at 37 °C ± 1 °C for 24 h ± 3 h

4.4 Plating out and identification

From the cultures obtained in 4.3, two selective solid media are inoculated:

 xylose lysine deoxycholate agar (XLD agar);

 any other solid selective medium complementary to XLD agar and especially appropriate for the isolation of

lactose-positive Salmonella and Salmonella Typhi and Salmonella Paratyphi strains; the laboratory may

choose which medium to use

The XLD agar is incubated at 37 °C ± 1 °C and examined after 24 h ± 3 h The second selective agar is incubated according to the manufacturer's recommendations

NOTE For information, Brilliant green agar (BGA), bismuth sulfite agar, etc., could be used as the second plating-out medium

4.5 Confirmation of identity

Colonies of presumptive Salmonella are subcultured, then plated out as described in 4.4, and their identity is

confirmed by means of appropriate biochemical and serological tests

5 Culture media, reagents and sera

5.1 General

For current laboratory practice, see ISO 7218

5.2 Culture media and reagents

NOTE Because of the large number of culture media and reagents, it is considered preferable, for clarity, to give their compositions and preparations in annex B

5.2.1 Non-selective pre-enrichment medium: Buffered peptone water

5.2.4 Solid selective plating-out media

5.2.4.1 First medium: Xylose lysine deoxycholate agar (XLD agar)

5.2.10 Reagents for Voges-Proskauer (VP) reaction

Every attempt should be made to ensure that the anti-sera used are adequate to provide for the detection of all

Salmonella serotypes Assistance towards this objective may be obtained by using only anti-sera prepared by a

supplier recognized as competent (for example, by an appropriate government agency)

6 Apparatus and glassware

Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications

Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following

6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave)

See ISO 7218

6.2 Drying cabinet or oven, ventilated by convection, capable of operating between 37 °C and 55 °C

6.3 Incubator, capable of operating at 37 °C ± 1 °C

6.4 Water bath, capable of operating at 41,5 °C ± 1 °C, or incubator, capable of operating at 41,5 °C ± 1 °C 6.5 Water baths, capable of operating at 44 °C to 47 °C

6.6 Water bath, capable of operating at 37 °C ± 1 °C

It is recommended to use a water bath (6.4, 6.5 and 6.6) containing an antibacterial agent because of the low

infective dose of Salmonella

6.7 Sterile loops, of diameter approximately 3 mm or 10 µl, or sterile pipettes

6.8 pH-meter, having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C

6.9 Test tubes or flasks, of appropriate capacity

Bottles or flasks with non-toxic metallic or plastic screw-caps may be used

6.10 Graduated pipettes or automatic pipettes, of nominal capacities 10 ml and 1 ml, graduated respectively in

8 Preparation of test sample

Prepare the test sample in accordance with the specific International Standard dealing with the product concerned

If there is no specific International Standard, it is recommended that the parties concerned come to an agreement

on this subject

9.1 Test portion and initial suspension

9.1.2 Specific preparations of the initial suspension for certain foodstuffs

NOTE The following specific preparations concern only the case of Salmonella Specific preparations applicable for the

determination of any microorganisms are described in ISO 6887-2, ISO 6887-3, ISO 6887-4 and ISO 8261

9.1.2.1 Cocoa and cocoa-containing products (e.g more than 20 %)

Add to the buffered peptone water (5.2.1) preferably 50 g/l of casein (avoid the use of acid casein), or 100 g/l of sterile skim milk powder and add, after about 2 h incubation, 0,018 g/l of Brilliant green if the foodstuff is likely to be highly contaminated with Gram-positive flora

9.1.2.2 Acidic and acidifying foodstuffs

Ensure that the pH does not fall to below 4,5 during pre-enrichment

NOTE The pH of acidic and acidifying foodstuffs is more stable if double-strength buffered peptone water is used

9.2 Non-selective pre-enrichment

Incubate the initial suspension (9.1) at 37 °C ± 1 °C for 18 h ± 2 h

9.3 Selective enrichment

9.3.1 Transfer 0,1 ml of the culture obtained in 9.2 to a tube containing 10 ml of the RVS broth (5.2.2); transfer

1 ml of the culture obtained in 9.2 to a tube containing 10 ml of MKTTn broth (5.2.3)

9.3.2 Incubate the inoculated RVS broth (9.3.1) at 41,5 °C ± 1 °C for 24 h ± 3 h and the inoculated MKTTn broth

at 37 °C ± 1 °C for 24 h ± 3 h Care should be taken that the maximum allowed incubation temperature (42,5 °C) is not exceeded

9.4 Plating out and identification

9.4.1 After incubation for 24 h ± 3 h, using the culture obtained in the RVS broth (9.3.2), inoculate by means of a

loop (6.7) the surface of one large-size Petri dish (6.11) containing the first selective plating-out medium (XLD agar, see 5.2.4.1), so that well-isolated colonies will be obtained

In the absence of large dishes, use two small dishes one after the other, using the same loop

Proceed in the same way with the second selective plating-out medium (5.2.4.2) using a sterile loop and Petri dishes as above

9.4.2 After incubation for 24 h ± 3 h, using the culture obtained in the MKTTn broth (9.3.2), repeat the procedure

described in 9.4.1 with the two selective plating-out media

9.4.3 Invert the dishes (9.4.1 and 9.4.2) so that the bottom is uppermost, and place them in the incubator (6.3)

set at 37 °C for the first plating-out medium (5.2.4.1) The manufacturer's instructions shall be followed for the second plating-out medium (5.2.4.2)

9.4.4 After incubation for 24 h ± 3 h, examine the plates (9.4.3) for the presence of typical colonies of Salmonella

and atypical colonies that may be Salmonella (see Note) Mark their position on the bottom of the dish

Typical colonies of Salmonella grown on XLD agar have a black centre and a lightly transparent zone of reddish

colour due to the colour change of the indicator

NOTE Salmonella H2S negative variants (e.g S Paratyphi A) grown on XLD agar are pink with a darker pink centre Lactose-positive Salmonella grown on XLD agar are yellow with or without blackening

Incubate the second selective solid medium at the appropriate temperature and examine after the appropriate time

to check for the presence of colonies which, from their characteristics, are considered to be presumptive

Salmonella

9.5 Confirmation

9.5.1 General

If shown to be reliable, commercially available identification kits for the biochemical examination of Salmonella may

be used The use of identification kits concerns the biochemical confirmation of colonies These kits should be used following the manufacturer's instructions

NOTE The recognition of colonies of Salmonella is to a large extent a matter of experience, and their appearance may vary

somewhat, not only from serovar to serovar, but also from batch to batch of the selective culture medium used

9.5.2 Selection of colonies for confirmation

For confirmation, take from each dish (two small-sized dishes or one large-sized dish) of each selective medium (see 9.4) at least one colony considered to be typical or suspect and a further four colonies if the first is negative

It is recommended that at least five colonies be identified in the case of epidemiological studies If on one dish there are fewer than five typical or suspect colonies, take for confirmation all the typical or suspect colonies

Streak the selected colonies onto the surface of pre-dried nutrient agar plates (5.2.5), in a manner which will allow well-isolated colonies to develop Incubate the inoculated plates (9.4.3) at 37 °C ± 1 °C for 24 h ± 3 h

Use pure cultures for biochemical and serological confirmation

Streak the agar slant surface and stab the butt Incubate at 37 °C ± 1 °C for 24 h ± 3 h

Interpret the changes in the medium as follows

a) Butt

 yellow glucose positive (glucose used)

 red or unchanged glucose negative (glucose not used)

 black formation of hydrogen sulfide

 bubbles or cracks gas formation from glucose

b) Slant surface

 yellow lactose and/or sucrose positive (lactose and/or sucrose used)

 red or unchanged lactose and sucrose negative (neither lactose nor sucrose used)

Typical Salmonella cultures show alkaline (red) slants and acid (yellow) butts with gas formation (bubbles) and (in

about 90 % of the cases) formation of hydrogen sulfide (blackening of the agar) (9.5.3.8)

When a lactose-positive Salmonella is isolated (see 4.4), the TSI slant is yellow Thus, preliminary confirmation of

Salmonella cultures shall not be based on the results of the TSI agar test only (see 9.5.3)

9.5.3.3 Urea agar (5.2.7)

Streak the agar slant surface Incubate at 37 °C ± 1 °C for 24 h ± 3 h and examine at intervals

If the reaction is positive, splitting of urea liberates ammonia, which changes the colour of phenol red to rose-pink and later to deep cerise The reaction is often apparent after 2 h to 4 h

9.5.3.4 L-Lysine decarboxylation medium (5.2.8)

Inoculate just below the surface of the liquid medium Incubate at 37 °C ± 1 °C for 24 h ± 3 h

Turbidity and a purple colour after incubation indicates a positive reaction A yellow colour indicates a negative reaction

9.5.3.5 Detection of β-galactosidase (5.2.9)

Suspend a loopful of the suspected colony in a tube containing 0,25 ml of the saline solution (5.2.13)

Add 1 drop of toluene and shake the tube Put the tube in a water bath (6.6) set at 37 °C and leave for several minutes (approximately 5 min) Add 0,25 ml of the reagent for detection of β-galactosidase and mix

Replace the tube in the water bath set at 37 °C and leave for 24 h ± 3 h, examining the tube at intervals

A yellow colour indicates a positive reaction The reaction is often apparent after 20 min

If prepared paper discs (5.2.9) are used, follow the manufacturer's instructions

9.5.3.6 Medium for Voges-Proskauer (VP) reaction (5.2.10)

Suspend a loopful of the suspected colony in a sterile tube containing 3 ml of the VP medium

Incubate at 37 °C ± 1 °C for 24 h ± 3 h

After incubation, add two drops of the creatine solution, three drops of the ethanolic solution of 1-naphthol and then two drops of the potassium hydroxide solution; shake after the addition of each reagent

The formation of a pink to bright red colour within 15 min indicates a positive reaction

9.5.3.7 Medium for indole reaction (5.2.11)

Inoculate a tube containing 5 ml of the tryptone/tryptophan medium with the suspected colony

Incubate at 37 °C ± 1 °C for 24 h ± 3 h After incubation, add 1 ml of the Kovacs reagent

The formation of a red ring indicates a positive reaction A yellow-brown ring indicates a negative reaction

9.5.3.8 Interpretation of the biochemical tests

Salmonella generally show the reactions given in Table 1

9.5.4 Serological confirmation and serotyping

9.5.4.1 General

The detection of the presence of Salmonella O-, Vi- and H-antigens is tested by slide agglutination with the

appropriate sera, from pure colonies (9.5.2) and after auto-agglutinable strains have been eliminated Use the antisera according to the producer's instructions if different from the description below

9.5.4.2 Elimination of auto-agglutinable strains

Place one drop of the saline solution (5.2.13) onto a carefully cleaned glass slide Disperse in the drop, by means

of a loop (6.7), part of the colony to be tested, in order to obtain a homogeneous and turbid suspension

NOTE It is also possible to disperse part of the colony to be tested in a drop of water, and then to mix this solution with one drop of saline solution (5.2.13)

Rock the slide gently for 30 s to 60 s Observe the result against a dark background, preferably with the aid of a magnifying glass

If the bacteria have clumped into more or less distinct units, the strain is considered auto-agglutinable, and shall not be submitted to the following tests as the detection of the antigens is not feasible

Table 1 — Interpretation of biochemical tests

Salmonella strain

Testa (9.5.3.2 to 9.5.3.7)

Reaction % b Reaction % b Reaction % c Reaction % c Reaction % b

b These percentages indicate that not all isolates of Salmonella serotype show the reactions marked + or − These percentages may vary

between and within serotypes of food poisoning serotypes from different locations

c The percentages are not known from available literature

d Salmonella Typhi is anaerogenic

e The Salmonella enterica subspecies arizonæ gives a positive or negative lactose reaction but is always β-galactosidase positive For the study of these strains it may be useful to carry out complementary tests

9.5.4.3 Examination for O-antigens

Using one non-autoagglutinating pure colony, proceed according to 9.5.4.2, using one drop of the anti-O serum (5.3) instead of the saline solution (5.2.13)

If agglutination occurs, the reaction is considered positive

Use the poly- and monovalent sera one after the other

9.5.4.4 Examination for Vi-antigens

Proceed according to 9.5.4.2, but using one drop of the anti-Vi serum (5.3) instead of the saline solution

If agglutination occurs, the reaction is considered positive

9.5.4.5 Examination for H-antigens

Inoculate the semi-solid nutrient agar (5.2.12) with a pure non-auto-agglutinable colony Incubate the medium at

37 °C ± 1 °C for 24 h ± 3 h

Use this culture for examination for the H-antigens, proceeding according to 9.5.4.2, but using one drop of the anti-H serum (5.3) instead of the saline solution

If agglutination occurs, the reaction is considered positive

9.5.5 Interpretation of biochemical and serological reactions

Table 2 gives the interpretation of the confirmatory tests (9.5.3 and 9.5.4) carried out on the colonies used (9.5.2)

Table 2 — Interpretation of confirmatory tests Biochemical reactions Auto-agglutination Serological reactions Interpretation

Salmonella

In accordance with the results of the interpretation, indicate the presence or absence of Salmonella in a test portion

of x g or x ml of product (see ISO 7218)

See annex C for the precision data obtained from the interlaboratory trial

11 Test report

The test report shall specify:

 the sampling method used, if known;

 any deviation in the enrichment media or the incubation conditions used;

 all operating conditions not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the results;

 the results obtained

The test report shall also state whether a positive result was obtained only when using a plating-out medium (5.2.4) not specified in this International Standard

12 Quality assurance

To check the ability of the laboratory to detect Salmonella with the methods and media described in this

International Standard, introduce reference samples into control flasks of the pre-enrichment medium (see 5.2.1) Proceed with the control flasks as for the test cultures